Identification of the conformational changes associated with the activation of a class I GPCR Class I GPCRs ('rhodopsin-like'GPCRs) represent by far the largest GPCR subfamily containing 800 full-length human receptor proteins. Members of this receptor family are the target of an extraordinarily large number of clinically important drugs. Little is known about the nature of the conformational changes that convert GPCRs activated by diffusible ligands from their resting into their active states. To study the molecular mechanisms underlying the activation of this class of receptors, we have used the M3 muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR, as a model system. To monitor ligand-induced changes in GPCR structure, we employed an in situ disulfide cross-linking strategy that allows the detection of disulfide bond formation between Cys residues that are adjacent to each other in the three-dimensional (3D) structure of the receptor. One major advantage of this strategy is that ligand-dependent conformational changes can be detected in receptors present in their native membrane environment, without the need for any receptor purification and reconstitution steps. We found that M3R activation was accompanied by structural changes involving multiple receptor regions, primarily distinct segments of transmembrane helices 3, 6, and 7, as well as helix 8 (Wess et al., Trends Pharmacol Sci 29, 616-625, 2008). Given the high degree of structural homology found among most GPCRs, it is likely that these findings will be of considerable general relevance. A better understanding of the molecular mechanisms underlying GPCR activation may lead to novel strategies aimed at modulating GPCR function for therapeutic purposes. Structural characterization of a GPCR/G protein interface The interaction of GPCRs with heterotrimeric G proteins represents one of the most fundamental processes regulating cell function. Although detailed structural information is now available for several GPCRs and heterotrimeric G proteins, the molecular architecture of the GPCR/G protein complex remains poorly defined, primarily due to the lack of a high-resolution X-ray structure. During the past year, we applied a comprehensive GPCR/G-alpha cross-linking strategy to map a receptor/G-alpha interface, both prior to and after agonist-induced receptor activation. By employing the M3R/GG-alpha-q system as a model system, we examined the ability of 250 combinations of Cys-substituted M3R and GG-alpha-q proteins to undergo cross-link formation. We identified many specific M3R/G-alpha-q contact sites, both in the inactive and the active receptor conformation. The observed M3R/G-alpha-q cross-linking patterns yielded a sufficient number of structural constraints allowing us to generate a model of the M3R/G-alpha-q complex. Since heterotrimeric G proteins as well as most GPCRs share a high degree of structural homology, our findings should be of great general relevance. Use of yeast expression technology to identify novel M3R-interacting proteins GPCRs do not function in isolation but are part of an intricate network of receptor-protein interactions that are responsible for various functions such as trafficking and targeting of the receptor to the membrane surface, stabilization of the receptor at the cell surface, and fine-tuning of the receptors pharmacology. One of our goals is to identify proteins that can interact with and modulate the function of the M3R. Such proteins may represent attractive novel targets for the treatment of various pathophysiological conditions including obesity and type 2 diabetes (Wess et al. Nat Rev Drug Discov 6, 721-33, 2007). In the past, many GPCR-interacting proteins have been identified by the use of classical yeast two-hybrid screening techniques. Usually, this approach requires that both interacting proteins are expressed, in a soluble form, in the nucleus. As a result, these screens cannot be applied to full-length GPCRs which are localized to cellular membranes. To overcome the limitations associated with the use of traditional yeast two-hybrid screening approaches, we employed the split ubiquitin membrane-based yeast two-hybrid system (Thaminy et al., Meth Mol Biol 261, 297-312, 2004) to screen for M3R-interacting proteins. The main advantage of this system, as compared to traditional yeast two-hybrid screening approaches, is that the bait protein (the full-length M3R or any other GPCR) is localized to the plasma membrane (or other cellular membranes) and proteins that are able to interact with the bait GPCR are identified by the use of simple growth and colorimetric assays. Initially, we expressed the M3R in yeast where it served as a bait to screen a human brain cDNA library for M3R interacting proteins (the M3R is widely expressed throughout the brain). The subsequent split ubiquitin yeast two-hybrid screen yielded >50 individual proteins that were able to interact with the M3R in a specific fashion in yeast;most of these interactions have not been identified previously. We recently confirmed the specificity of several of these interactions in a mammalian expression system. Studies are presently underway to determine the functional roles of the newly identified M3R-interacting proteins in mammalian cells. Among several other techniques, we are employing siRNA technology to knock down the expression of specific M3R-associated proteins in neuronal cells and in beta-cell lines that express endogenous M3Rs. These studies should reveal the role of specific M3R-associated proteins in M3R function. Given the lack of ligands that can regulate M3R activity with high selectivity, we speculate that the functional characterization of these newly identified M3R-associated proteins may suggest new strategies to modulate M3R function for therapeutic purposes.